Method and medicament for inhibiting the expression of a given gene

ABSTRACT

The present invention provides methods and compositions for inhibiting gene expression using double stranded RNA molecules that are between 15 and 21 nucleotides in length and are complementary to a target gene sequence.

The present application is a divisional application of U.S. applicationSer. No. 09/889,802, filed Sep. 17, 2001, which is the National Stage ofInternational Application No. PCT/DE00/00244, filed Jan. 29, 2000, andclaims priority to DE19903713.2, filed Jan. 30, 1999 and DE19956568.6,tiled Nov. 24, 1999. The contents of all these applications areincorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

Not applicable.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention is in the field of inhibition of gene expressionusing double stranded oligoribonuclcotides.

Methods of inhibiting gene expression using double strandedoligoribonucleotides have recently been described.

Such a method is known from WO 99/32619, which was unpublished at thepriority date of the present invention. The known process aims atinhibiting the expression of genes in cells of invertebrates. To thisend, the double-stranded oligoribonucleotide must exhibit a sequencewhich is identical with the target gene and which has a length of atleast 50 bases. To achieve efficient inhibition, the identical sequencemust be 300 to 1 000 base pairs in length. Such an oligoribonucleotideis complicated to prepare.

DE 196 31 919 C2 describes an antisense RNA with specific secondarystructures, the antisense RNA being present in the form of a vectorencoding it. The antisense RNA takes the form of an RNA molecule whichis complementary to regions of the mRNA. Inhibition of the geneexpression is caused by binding to these regions. This inhibition can beemployed in particular for the diagnosis and/or therapy of diseases, forexample tumor diseases or viral infections.—The disadvantage is that theantisense RNA must be introduced into the cell in an amount which is atleast as high as the amount of the mRNA. The known antisense methods arenot particularly effective.

U.S. Pat. No. 5,712,257 discloses a medicament comprising mismatcheddouble-stranded RNA (dsRNA) and bioactive mismatched fragments of dsRNAin the form of a ternary complex together with a surfactant. The dsRNAused for this purpose consists of synthetic nucleic acid single strandswithout defined base sequence. The single strands undergo irregular basepairing, also known as “non-Watson-Crick” base pairing, giving rise tomismatched double strands. The known dsRNA is used to inhibit theamplification of retroviruses such as HIV. Amplification of the viruscan be inhibited when non-sequence-specific dsRNA is introduced into thecells. This leads to the induction of interferon, which is intended toinhibit viral amplification. The inhibitory effect, or the activity, ofthis method is poor.

It is known from Fire, A. et al., NATURE, Vol. 391, pp. 806 that dsRNAwhose one strand is complementary in segments to a nematode gene to beinhibited inhibits the expression of this gene highly efficiently. It isbelieved that the particular activity of the dsRNA used in nematodecells is not due to the antisense principle but possibly on catalyticproperties of the dsRNA, or enzymes induced by it.—Nothing is mentionedin this paper on the activity of specific dsRNA with regard toinhibiting the gene expression, in particular in mammalian and humancells.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods and compositions for inhibitinggene expression using double stranded RNA molecules as well as doublestranded RNA molecules containing modified bases.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the schematic representation of a plasmid for the in vitrotranscription with T7- and SP6-polymerase.

FIG. 2 shows RNA following electrophoresis on an 8% polyacrylamide geland staining with ethidium bromide.

FIG. 3 shows a representation of radioactive RNA transcripts followingelectrophoresis on an 8% polyacrylamide gel with 7M urea by means of aninstant imager.

FIG. 4 a-e shows Texas Red and YFP fluorescence in murine fibroblasts.

DETAILED DESCRIPTION OF THE INVENTION

The object of the present invention is to do away with the disadvantagesof the prior art. In particular, it is intended to provide as effectiveas possible a method, medicament or use for the preparation of amedicament, which method, medicament or use is capable of causingparticularly effective inhibition of the expression of a given targetgene.

This object is achieved by the features of the claims presented below.Advantageous embodiments can be seen from these claims.

In accordance with the method-oriented inventions, it is provided ineach case that the region I which is complementary to the target geneexhibits not more than 49 successive nucleotide pairs.

Provided in accordance with the invention are an oligoribonucleotide ora vector encoding therefor. At least segments of the oligoribonucleotideexhibit a defined nucleotide sequence. The defined segment may belimited to the complementary region I. However, it is also possible thatall of the double-stranded oligoribonucleotide exhibits a definednucleotide sequence.

Surprisingly, it has emerged that an effective inhibition of theexpression of the target gene can be achieved even when thecomplementary region I is not more than 49 base pairs in length. Theprocedure of providing such oligoribonucleotides is less complicated.

In particular, dsRNA with a length of over 50 nucleotide pairs inducescertain cellular mechanisms, for example the dsRNA-dependent proteinkinase or the 2-5A system, in mammalian and human cells. This leads tothe disappearance of the interference effect mediated by the dsRNA whichexhibits a defined sequence. As a consequence, protein biosynthesis inthe cell is blocked. The present invention overcomes this disadvantagein particular.

Furthermore, the uptake of dsRNA with short chain lengths into the cellor into the nucleus is facilitated markedly over longer-chain dsRNAs.

It has proved advantageous for the dsRNA or the vector to be presentpackaged into micellar structures, preferably in liposomes. The dsRNA orthe vector can likewise be enclosed in viral natural capsids or inchemically or enzymatically produced artificial capsids or structuresderived therefrom.—The above-mentioned features make it possible tointroduce the dsRNA or the vector into given target cells.

In a further aspect, the dsRNA has 10 to 1 000, preferably 15 to 49,base pairs. Thus, the dsRNA can be longer than the region I, which iscomplementary to the target gene. The complementary region I can belocated at the terminus or inserted into the dsRNA. Such dsRNA or avector provided for coding the same can be produced synthetically orenzymatically by customary methods.

The gene to be inhibited is expediently expressed in eukaryotic cells.The target gene can be selected from the following group: oncogene,cytokin gene, Id protein gene, developmental gene, prion gene. It canalso be expressed in pathogenic organisms, preferably in plasmodia. Itcan be part of a virus or viroid which is preferably pathogenic tohumans.—The method proposed makes it possible to produce compositionsfor the therapy of genetically determined diseases, for example cancer,viral diseases or Alzheimer's disease.

The virus or viroid can also be a virus or viroid which is pathogenic toanimals or plant-pathogenic. In this case, the method according to theinvention also permits the provision of compositions for treating animalor plant diseases.

In a further aspect, segments of the dsRNA are designed asdouble-stranded. A region II which is complementary within thedouble-stranded structure is formed by two separate RNA single strandsor by autocomplementary regions of a topologically closed RNA singlestrand which is preferably in circular form.

The ends of the dsRNA can be modified to counteract degradation in thecell or dissociation into the single strands. Dissociation takes placein particular when low concentrations or short chain lengths are used.To inhibit dissociation in a particularly effective fashion, thecohesion of the complementary region II, which is caused by thenucleotide pairs, can be increased by at least one, preferably two,further chemical linkage(s).—A dsRNA according to the invention whosedissociation is reduced exhibits greater stability to enzymatic andchemical degradation in the cell or in the organism.

The complementary region. II can be formed by autocomplementary regionsof an RNA hairpin loop, in particular when using a vector according tothe invention. To afford protection from degradation, it is expedientfor the nucleotides to be chemically modified in the loop region betweenthe double-stranded structure.

The chemical linkage is expediently formed by a covalent or ionic bond,a hydrogen bond, hydrophobic interactions, preferably van-der-Waals orstacking interactions, or by metal-ion coordination. In an especiallyadvantageous aspect, it can be formed at at least one, preferably both,end(s) of the complementary region II.

It has furthermore proved to be advantageous for the chemical linkage tobe formed by one or more linkage groups, the linkage groups preferablybeing poly(oxyphosphinicooxy-1,3-propanediol) and/or poly-ethyleneglycol chains. The chemical linkage can also be formed by purine analogsused in place of purines in the complementary regions II. It is alsoadvantageous for the chemical linkage to be formed by azabenzene unitsintroduced into the complementary regions II. Moreover, it can be formedby branched nucleotide analogs used in place of nucleotides in thecomplementary regions II.

It has proved expedient to use at least one of the following groups forgenerating the chemical linkage: methylene blue; bifunctional groups,preferably bis(2-chloroethyl)amine;N-acetyl-N′-(p-glyoxyl-benzoyl)cystamine; 4-thiouracil; psoralene. Thechemical linkage can furthermore be formed by thiophosphoryl groupsprovided at the ends of the double-stranded region. The chemical linkageat the ends of the double-stranded region is preferably formed bytriple-helix bonds.

The chemical linkage can expediently be induced by ultraviolet light.

The nucleotides of the dsRNA can be modified. This counteracts theactivation, in the cell, of a double-stranded-RNA-dependent proteinkinase, PKR. Advantageously, at least one 2′-hydroxyl group of thenucleotides of the dsRNA in the complementary region II is replaced by achemical group, preferably a 2′-amino or a 2′-methyl group. At least onenucleotide in at least one strand of the complementary region II canalso be a locked nucleotide with a sugar ring which is chemicallymodified, preferably by a 2′-O, 4′-C methylene bridge. Advantageously,several nucleotides are locked nucleotides.

A further especially advantageous embodiment provides that the dsRNA orthe vector is bound to, associated with or surrounded by, at least oneviral coat protein which originates from a virus, is derived therefromor has been prepared synthetically. The coat protein can be derived frompolyomavitus. The coat protein can contain the polyomavirus virusprotein 1 (VP1) and/or virus protein 2 (VP2). The use of such coatproteins is known from, for example, DE 196 18 797 A1, whose disclosureis herewith incorporated.—The abovementioned features considerablyfacilitate the introduction of the dsRNA or of the vector into the cell.

When a capsid or capsid-type structure is formed from the coat protein,one side preferably faces the interior of the capsid or capsid-typestructure. The construct formed is particularly stable.

The dsRNA can be complementary to the primary or processed RNAtranscript of the target gene.—The cell can be a vertebrate cell or ahuman cell.

At least two dsRNAs which differ from each other or at least one vectorencoding them can be introduced into the cell, where at least segmentsof one strand of each dsRNA are complementary to in each case one of atleast two different target genes. This makes it possible simultaneouslyto inhibit the expression of at least two different target genes. Inorder to suppress, in the cell, the expression of adouble-stranded-RNA-dependent protein kinase, PKR, one of the targetgenes is advantageously the PKR gene. This allows effective suppressionof the PKR activity in the cell.

The invention furthermore provides a medicament with at least oneoligoribonucleotide with double-stranded structure (dsRNA) forinhibiting the expression of a given target gene, where one strand ofthe dsRNA has a region I where at least segments are complementary tothe target gene.—Surprisingly, it has emerged that such a dsRNA issuitable as medicament for inhibiting the expression of a given gene inmammalian cells. In comparison with the use of single-strandedoligoribonucleotides, the inhibition is already caused at concentrationswhich are lower by at least one order of magnitude. The medicamentaccording to the invention is highly effective. Lesser side effects canbe expected.

The invention furthermore provides a medicament with at least one vectorfor coding at least one oligoribonucleotide with double-strandedstructure (dsRNA) for inhibiting the expression of a given target gene,where one strand of the dsRNA has a region I where at least segments arecomplementary to the target gene.—The medicament proposed exhibits theabovementioned advantages. By using a vector, in particular productioncosts can be reduced.

In a particularly advantageous embodiment, the complementary region Ihas not more than 49 successive nucleotide pairs.—Surprisingly, it hasemerged that an effective inhibition of the expression of the targetgene can be achieved even when the complementary region I is not morethan 49 base pairs in length. The procedure of providing sucholigoribonucleotides is less complicated.

The invention furthermore provides a use of an oligoribonucleotide withdouble-stranded structure (dsRNA) for preparing a medicament forinhibiting the expression of a given target gene, where one strand ofthe dsRNA has a region I where at least segments are complementary tothe target gene.—Surprisingly, such a dsRNA is suitable for preparing amedicament for inhibiting the expression of a given gene. Compared withthe use of single-stranded oligoribonucleotides, the inhibition isalready caused at concentrations which are lower by one order ofmagnitude when using dsRNA. The use according to the invention thusmakes possible the preparation of particularly effective medicaments.

The invention furthermore provides the use of a vector for coding atleast one oligoribonucleotide with double-stranded structure (dsRNA) forpreparing a medicament for inhibiting the expression of a given targetgene, where one strand of the dsRNA has a region I where at leastsegments are complementary to this target gene.—The use of a vectormakes possible a particularly effective gene therapy.

With regard to advantageous embodiments of the medicament and of theuse, reference is made to the description of the above features.

Use examples of the invention are illustrated in greater detailhereinbelow with reference to the figures, in which:

USE EXAMPLE 1

The inhibition of transcription was detected by means of sequencehomologous dsRNA in an in vitro transcription system with a nuclearextract from human HeLa cells. The DNA template for this experiment wasplasmid pCMV1200 which had been linearized by means of BamHI.

Generation of the Template Plasmids:

The plasmid shown in FIG. 1 was constructed for use in the enzymaticsynthesis of the dsRNA. To this end, a polymerase chain reaction (PCR)with the “positive control DNA” of the HelaScribe® Nuclear Extract invitro transcription kit by Promega, Madison, USA, as DNA template wasfirst carried out. One of the primers used contained the sequence of anEcoRI cleavage site and of the T7 RNA polymerase promoter as shown insequence listing No. 1. The other primer contained the sequence of aBamHI cleavage site and of the SP6 RNA polymerase promoter as shown insequence listing No. 2. In addition, the two primers had, at the 3′ends, regions which were identical with or complementary to the DNAtemplate. The PCR was carried out by means of the “Taq PCR Core Kits” byQiagen, Hilden, Germany, following the manufacturer's instructions. 1.5mM MgCl₂, in each case 200 μM dNTP, in each case 0.5 μM primer, 2.5 UTag DNA polymerase and approximately 100 ng of “positive control DNA”were employed as template in PCR buffer in a volume of 100 μl. Afterinitial denaturation of the template DNA by heating for 5 minutes at 94°C., amplification was carried out in 30 cycles of denaturation for ineach case 60 seconds at 94° C., annealing for 60 seconds at 5° C. belowthe calculated melting point of the primers and polymerization for 1.5-2minutes at 72° C. After a final polymerization of 5 minutes at 72° C., 5μl of the reaction were analyzed by agarose-gel electrophoresis. Thelength of the DNA fragment amplified thus was 400 base pairs, 340 basepairs corresponding to the “positive control DNA”. The PCR product waspurified, hydrolyzed with EcoRI and BamHI and, after repurification,employed in the ligation together with a pUC18 vector which had alsobeen hydrolyzed by EcoRI and BamHI. E. coli XL1-blue was thentransformed. The plasmid obtained (pCMV5) carries a DNA fragment whose5′ end is flanked by the T7 promoter and whose 3′ end is flanked by theSP6 promoter. By linearizing the plasmid with BamHI., it can be employedin vitro with the T7-RNA polymerase for the run-off transcription of asingle-stranded RNA which is 340 nucleotides in Length and shown insequence listing No. 3. If the plasmid is linearized with EcoRI, it canbe employed for the run-off transcription with SP6 RNA polymerase,giving rise to the complementary strand. In accordance with the methodoutlined hereinabove, an RNA 23 nucleotides in length was alsosynthesized. To this end, a DNA shown in sequence listing No. 4 wasligated with the pUC18 vector via the EcoRI and BamHI cleavage sites.

Plasmid pCMV1200 was constructed as DNA template for the in-vitrotranscription with HeLa nuclear extract. To this end, a 1 191 bpEcoRI/BamHI fragment of the positive control DNA contained in theHeLaScribe® Nuclear Extract in vitro transcription kit was amplified bymeans of PCR. The amplified fragment encompasses the 828 bp “immediateearly” CMV promoter and a 363 bp transcribable DNA fragment. The PCRproduct was ligated to the vector pGEM-T via “T-overhang” ligation. ABamHI cleavage site is located at the 5′ end of the fragment. Theplasmid was linearized by hydrolysis with BamHI and used as template inthe run-off transcription.

In-Vitro Transcription of the Complementary Single Strands:

pCMV5 plasmid DNA was linearized with EcoRI or BamHI. It was used as DNAtemplate for an in-vitro transcription of the complementary RNA singlestrands with SP6 and T7 RNA polymerase, respectively. The “Riboprobe invitro Transcription” system by Promega, Madison, USA, was employed forthis purpose. Following the manufacturer's instructions, 2 μg oflinearized plasmid DNA were incubated in 100 μl of transcription bufferand 40 U T7 or SP6 RNA polymerase for 5-6 hours at 37° C. The DNAtemplate was subsequently degraded by addition of 2.5 μl of RNase-freeDNase RQ1 and incubation for 30 minutes at 37° C. The transcriptionreaction was made up to 300 μl with H₂O and purified by phenolextraction. The RNA was precipitated by addition of 150 μl of 7 Mammonium acatate [sic] and 1 125 μl of ethanol and stored at −65° C.until used for the hybridization.

Generation of the RNA Double Strands:

For the hybridization, 500 μl of the single-stranded RNA which had beenstored in ethanol and precipitated were spun down. The resulting pelletwas dried and taken up in 30 μl of PIPES buffer, pH 6.4 in the presenceof 80% formamide, 400 mM NaCl and 1 mM EDTA. In each case 15 μl of thecomplementary single strands were combined and heated for 10 minutes at85° C. The reactions were subsequently incubated overnight at 50° C. andcooled to room temperature.

Only approximately equimolar amounts of the two single strands wereemployed in the hybridization. This is why the dsRNA preparationscontained single-stranded RNA (ssRNA) as contaminant. In order to removethese ssRNA contaminants, the reactions were treated, afterhybridization, with the single-strand-specific ribonucleases bovinepancreatic RNase A and Aspergillus oryzae RNase T1. RNase A is anendoribonuclease which is specific for pyrimidines. RNase T1 is anendoribonuclease which preferentially cleaves at the 3′ side ofguanosines. dsRNA is no substrate for these ribonucleases. For the RNasetreatment, the reactions in 300 μl of Tris, pH 7.4, 300 mM NaCl and 5 mMEDTA were treated with 1.2 μl of RNaseA at a concentration of 10 mg/mland 2 μl of RNaseT1 at a concentration of 290 μg/ml. The reactions wereincubated for 1.5 hours at 30° C. Thereupon, the RNases were denaturedby addition of 5 μl of proteinase K at a concentration of 20 mg/ml and10 μl of 20% SDS and incubation for 30 minutes at 37° C. The dsRNA waspurified by phenol extraction and precipitated with ethanol. To verifythe completeness of the RNase digestion, two control reactions weretreated with ssRNA analogously to the hybridization reactions.

The dried pellet was taken up in 15 μl of TE buffer, pH 6.5, andsubjected to native polyacrylamide gel electrophoresis on an 8% gel. Theacrylamide gel was subsequently stained in an ethidium bromide solutionand washed in a water bath. FIG. 2 shows the RNA which had beenvisualized in a UV transilluminator. The sense RNA which had beenapplied to lane 1 and the antisense RNA which had been applied to lane 2showed a different migration behavior under the chosen conditions thanthe dsRNA of the hybridization reaction which had been applied to lane3. The RNase-treated sense RNA and antisense RNA which had been appliedto lanes 4 and 5, respectively, produced no visible band. This showsthat the single-stranded RNAs had been degraded completely. TheRNase-treated dsRNA of the hybridization reaction which had been appliedto lane 6 is resistant to RNase treatment. The band which migratesfaster in the native gel in comparison with the dsRNA applied to lane 3results from dsRNA which is free from ssRNA. In addition to the dominantmain band, weaker bands which migrate faster are observed after theRNase treatment.

In-Vitro Transcription Test with Human Nuclear Extract:

Using the HeLaScribe® Nuclear Extract in vitro transcription kit byPromega, Madison, USA, the transcription efficiency of theabovementioned DNA fragment which is present in plasmid pCMV1200 andhomologous to the “positive control DNA” was determined in the presenceof the dsRNA (dsRNA-CMV5) with sequence homology. Also, the effect ofthe dsRNA without sequence homology, which corresponds to the yellowfluorescent protein (YFP) gene (dsRNA-YRP), was studied. This dsRNA hadbeen generated analogously to the dsRNA with sequence homology. Thesequence of a strand of this dsRNA can be found in sequence listing No.5. Plasmid pCMV1200 was used as template for the run-off transcription.It carries the “immediate early” cytomegalovirus promoter which isrecognized by the eukaryotic RNA polymerase II, and a transcribable DNAfragment. Transcription was carried out by means of the HeLa nuclearextract, which contains all the proteins which are necessary fortranscription. By addition of [•-³² P]rGTP to the transcriptionreaction, radiolabeled transcript was obtained. The [•-³²P]rGTP used hada specific activity of 400 Ci/mmol, 10 mCi/ml. 3 mM MgCl₂, in each case400 μM rATP, rCTP, rUTP, 16 μM rGTP, 0.4 μM [•-³²P]rGTP and depending onthe experiment 1 fmol of linearized plasmid DNA and various amounts ofdsRNA in transcription buffer were employed per reaction. Each batch wasmade up to a volume of 8.5 μl with H₂O. The reactions were mixedcarefully. To start the transcription, 4 U HeLa nuclear extract in avolume of 4 μl were added and incubated for 60 minutes at 30° C. Thereaction was stopped by addition of 87.5 μl of quench mix which had beenwarmed to 30° C. To remove the proteins, the reactions were treated with100 μl of phenol/chloroform/isoamyl alcohol (25:24:1 v/v/v) saturatedwith TE buffer, pH 5.0, and the reactions were mixed vigorously for 1minute. For phase separation, the reactions were spun for approximately1 minute at 12 000 rpm and the top phase was transferred into a freshreaction vessel. Each reaction was treated with 250 μl of ethanol. Thereactions were mixed thoroughly and incubated for at least 15 minutes ondry ice/methanol. To precipitate the RNA, the reactions were spun for 20minutes at 12 000 rpm and 40° C. The supernatant was discarded. Thepellet was dried in vacuo for 15 minutes and resuspended in 10 μl ofH₂O. Each reaction was treated with 10 μl of denaturing loading buffer.The free GTP was separated from the transcript formed by means ofdenaturing polyacrylamide gel electrophoresis on an 8% gel with 7 Murea. The RNA transcripts formed upon transcription with HeLa nuclearextract, in denaturing loading buffer, were heated for 10 minutes at 90°C. and 10 μl aliquots were applied immediately to the freshly washedpockets. The electrophoresis was run at 40 mA. The amount of theradioactive ssRNA formed upon transcription was analyzed afterelectrophoresis with the aid of an Instant Imager.

FIG. 3 shows the radioactive RNA from a representative test, shown bymeans of the Instant Imager. Samples obtained from the followingtranscription reactions were applied:

-   Lane 1: without template DNA, without dsRNA;-   Lane 1: 50 ng of template DNA, without dsRNA;-   Lane 3: 50 ng of template DNA, 0.5 μg of dsRNA YFP;-   Lane 4: 50 ng of template DNA, 1.5 μg of dsRNA YFP;-   Lane 5: 50 ng of template DNA, 3 μg of dsRNA YFP;-   Lane 6: 50 ng of template DNA, 5 μg of dsRNA YFP;-   Lane 7: without template DNA, 1.5 dsRNA YFP;-   Lane 8; 50 ng of template DNA, without dsRNA;-   Lane 9: 50 ng of template DNA, 0.5 μg of dsRNA CMV5;-   Lane 10: 50 ng of template DNA, 1.5 μg of dsRNA CMV5;-   Lane 11: 50 ng of template DNA, 3 μg of dsRNA CMV5;-   Lane 12: 50 ng of template DNA, 5 μg of dsRNA CNV5;

It emerged that the amount of transcript was reduced markedly in thepresence of dsRNA with sequence homology in comparison with the controlreaction without dsRNA and with the reactions with dsRNA YFP withoutsequence homology. The positive control in lane 2 shows that radioactivetranscript was formed upon the in-vitro transcription with HeLa nuclearextract. The reaction is used for comparison with the transcriptionreactions which had been incubated in the presence of dsRNA. Lanes 3 to6 show that the addition of non-sequentially-specific dsRNA YFP had noeffect on the amount of transcript formed. Lanes 9 to 12 show that theaddition of an amount of between 1.5 and 3 μg of sequentially-specificdsRNA CMV5 leads to a reduction in the amount of transcript formed. Inorder to exclude that the effects observed are based not on the dsRNAbut on any contamination which might have been carried alongaccidentally during the preparation of the dsRNA, a further control wascarried out. Single-stranded RNA was transcribed as described above andsubsequently subjected to the RNase treatment. It was demonstrated bymeans of native polyacrylamide gel electrophoresis that the ssRNA hadbeen degraded completely. This reaction was subjected to phenolextraction and ethanol precipitation and subsequently taken up in PEbuffer, as were the hybridization reactions. This gave a sample whichcontained no RNA but had been treated with the same enzymes and buffersas the dsRNA. Lane 8 shows that the addition of this sample had noeffect on transcription. The reduction of the transcript upon additionof sequence-specific dsRNA can therefore be ascribed unequivocally tothe dsRNA itself. The reduction of the amount of transcript of a gene inthe presence of dsRNA in a human transcription system indicates aninhibition of the expression of the gene in question. This effect can beattributed to a novel mechanism caused by the dsRNA.

USE EXAMPLE 2

The test system used for these in-vivo experiments was the murinefibroblast cell line NIH3T3, ATCC CRL-1658. The YFP gene was introducedinto the nuclei with the aid of microinjection. Expression of YFP wasstudied under the effect of simultaneously cotransfected dsRNA withsequence homology. This dsRNA YFP shows homology with the 5′-region ofthe YFP gene over a length of 315 bp. The nucleotide sequence of astrand of the dsRNA YRP is shown in sequence listing No. 5. Evaluationunder the fluorescence microscope was carried out 3 hours afterinjection with reference to the greenish-yellow fluorescence of the YFPformed.

Construction of the Template Plasmid, and Preparation of the dsRNA:

A plasmid was constructed following the same principle as described inuse example 1 to act as template for the production of the YFP dsRNA by,means of T7 and SP6 in-vitro transcription. Using the primer Eco_T7_YFPas shown in sequence listing No. 6 and Bam_SP6_YFP as shown in sequencelisting No. 7, the desired gene fragment was amplified by PCR and usedanalogously to the above description for preparing the dsRNA. The dsRNAYFP obtained is identical to the dsRNA used in use example 1 asnon-sequence-specific control.

A dsRNA linked chemically at the 3′ end of the RNA as shown in sequencelisting No. 8 to the 5′ end of the complementary RNA via a C18 linkergroup was prepared (L-dsRNA). To this end, synthons modified bydisulfide bridges were used. The 3′-terminal synthon is bound to thesolid support via the 3′ carbon with an aliphatic linker group via adisulfide bridge. In the 5′-terminal synthon of the complementaryoligoribonucleotide which is complementary to the 3′-terminal synthon ofthe one oligoribonucleotide, the 5′-trityl protecting group is bound viaa further aliphatic linker and a disulfide bridge. Following synthesisof the two single strands, removal of the protecting groups andhybridization of the complementary oligoribonucleotides, the thiolgroups which form are brought into spatial vicinity. The single strandsare linked to each other by oxidation via their aliphatic linkers and adisulfide bridge. This is followed by purification with the aid of HPLC.

Preparation of the Cell Cultures:

The cells were incubated in DMEM supplemented with 4.5 g/l glucose, 10%fetal bovine serum in culture dishes at 37° C. under a 7.5% CO₂atmosphere and passaged before reaching confluence. The cells weredetached with trypsin/EDTA. To prepare for microinjection, the cellswere transferred into Petri dishes and incubated further untilmicrocolonies formed.

Microinjection:

For the microinjection, the culture dishes were removed from theincubator for approximately 10 minutes. Approximately 50 nuclei wereinjected singly per reaction within a marked area using the AISmicroinjection system from Carl Zeiss, Göttingen, Germany. The cellswere subsequently incubated for three more hours. For themicroinjection, borosilicate glass capillaries from Hilgenberg GmbH,Malsfeld, Germany, with a diameter of less than 0.5 μm at the tip wereprepared. The microinjection was carried out using a micromanipulatorfrom Narishige Scientific Instrument Lab., Tokyo, Japan. The injectiontime was 0.8 seconds and the pressure was approximately 100 hPa. Thetransfection was carried out using the plasmid pCDNA YFP, which containsan approximately 800 bp BamHI/EcoRI fragment with the YFP gene in vectorpcDNA3. The samples injected into the nuclei contained 0.01 μg/μl ofpCDNA-YFP and Texas Red coupled to dextran-70000 in 14 mM NaCl, 3 mMKCl, 10 mM KPO₄ [sic], ph 7.5. Approximately 100 μl of RNA with aconcentration of 1 μM or, in the case of the L-dsRNA, 375 μM wereadditionally added.

The cells were studied under a fluorescence microscope with excitationwith the light of the excitation wavelength of Texas Red, 568 nm, or ofYFP, 488 nm. Individual cells were documented by means of a digitalcamers. FIGS. 4 a-e show the result for NIH3T3 cells. In the cells shownin FIG. 4 a, sense-YFP-ssRNA has been injected, in FIG. 4 bantisense-YFP-ssRNA, in FIG. 4 c dsRNA-YFP, in FIG. 4 d no RNA and inFIG. 4 e L-dsRNA.

The field on the left shows in each case the fluorescence of cells withexcitation at 568 nm. The fluorescence of the same cells at anexcitation of 488 nm is seen on the right. The Texas Red fluorescence ofall the cells shown demonstrates that the injection solution had beenapplied successfully into the nuclei and that cells with successful hitswere still alive after three hours. Dead cells no longer showed. TexasRed fluorescence.

The right fields of each of FIGS. 4 a and 4 b show that YFP expressionwas not visibly inhibited when the single-stranded RNA was injected intothe nuclei. The right field of FIG. 4 c shows cells whose YFPfluorescence was no longer detectable after the injection of dsRNA-YFP.FIG. 4 d shows cells into which no RNA had been injected, as control.The cell shown in FIG. 4 e shows YFP fluorescence which can no longer bedetected owing to the injection of the L-dsRNA which shows regions withsequence homology to the YFP gene. This result demonstrates that evenshorter dsRNAs can be used for specifically inhibiting gene expressionin mammals when the double strands are stabilized by chemically linkingthe single strands.

Literatur:

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1. An isolated oligoribonucleotide consisting of two separatecomplementary RNA single strands comprising a first strand and a secondstrand that are chemically linked, forming a double-stranded structure(dsRNA), wherein the dsRNA is 21 base pairs in length, wherein the dsRNAdoes not comprise a full length RNA transcript of a mammalian targetgene, wherein one strand of said strands of the dsRNA is complementaryto less than the full length of a third strand, said third strandcomprising an RNA transcript of said mammalian target gene, and whereinan amount of said third strand is capable of being reduced in responseto said dsRNA being introduced into the presence of said third strand ina mammalian cell, and wherein the dsRNA is capable of specificallyinhibiting the expression of said mammalian target gene usingdsRNA-mediated interference.
 2. The dsRNA of claim 1, wherein saidchemical linkage is formed by a covalent bond or hydrogen bond.
 3. ThedsRNA of claim 1, wherein said chemical linkage is a covalent linkage.4. The dsRNA of claim 3, wherein said covalent linkage comprises a C18linker group.
 5. The dsRNA of claim 1, wherein said chemical linkage isa labile linkage.
 6. The dsRNA of claim 5, wherein said labile linkagecomprises a disulfide bridge.
 7. The dsRNA of claim 1, wherein saidchemical linkage comprises a covalent linkage that is labile.
 8. ThedsRNA of claim 1, wherein the RNA transcript is a primary or a processedRNA.
 9. The dsRNA of claim 1, wherein said two separate complementarystrands are fully complementary to each other.
 10. The dsRNA of claim 1,wherein one of the single strands is complementary to the other of thesingle strands, wherein the two separate single strands are hybridizedto each other to form the double-stranded structure, and wherein the oneof the single strands is also chemically linked to the other of thesingle strands.
 11. The dsRNA of claim 1, wherein the dsRNA is capableof specifically inhibiting the expression at a concentration that islower by one order of magnitude than a concentration required for acorresponding single-stranded oligoribonucleotide to inhibit expression.12. The dsRNA of claim 1, wherein said dsRNA is non-autocomplementary.13. The dsRNA of claim 1, wherein said chemical linkage is acarbon-carbon linker.
 14. The dsRNA of claim 1, wherein saidchemically-linked separate complementary RNA single strands have reduceddissociation as compared to non-linked RNA strands.
 15. The dsRNA ofclaim 1, wherein said second and third strands are sense strands andwherein said first strand is an antisense strand.
 16. The dsRNA of claim1, wherein said first strand is capable of hybridizing to said thirdstrand to reduce the amount of said third strand.
 17. The dsRNA of claim1, wherein a 3′ end of one of said first and second strands is linked toa 5′ end of another of said first and second strands with acarbon-carbon linker.